Abstract

Shugoshins (Sgo) are conserved proteins that act as protectors of centromeric cohesion and as sensors of tension for the machinery that eliminates improper kinetochore-microtubule attachments. Most vertebrates contain two Sgo proteins, but their specific functions are not always clear. Xenopus laevis Sgo1, XSgo1, protects centromeric cohesin from the prophase dissociation pathway. Here, we report the identification of XSgo2 and show that it does not regulate cohesion. Instead, we find that it participates in bipolar spindle assembly. Both Sgo proteins interact physically with the Chromosomal Passenger Complex (CPC) containing Aurora B, a key regulator of mitosis, but the functional consequences of such interaction are distinct. XSgo1 is required for proper localization of the CPC while XSgo2 positively contributes to its activation and the subsequent phosphorylation of at least one key substrate for bipolar spindle assembly, the microtubule depolymerizing kinesin MCAK (Mitotic Centromere-Associated Kinesin). Thus, the two Xenopus Sgo proteins have non-overlapping functions in chromosome segregation. Our results further suggest that this functional specificity could rely on the association of XSgo1 and XSgo2 with different regulatory subunits of the PP2A complex.

Characterization of Xenopus Sgo2. (A) Schematic drawing of XSgo2 (top) and sequence alignment of the conserved coiled-coil and basic regions of Sgo proteins from the indicated species, including XSgo2 (bottom). Identical and similar amino acids are shown in red and blue, respectively. (B) Localization of XSgo1 (red) and XSgo2 (green) in interphase nuclei and mitotic chromosomes assembled from sperm chromatin in Xenopus egg extracts. (C) Replicated mitotic chromosomes assembled in mock-depleted extracts and extracts depleted of XSgo1 (Δ XSgo1), XSgo2 (Δ XSgo2), Bub1 (Δ Bub1) or Aurora B (Δ Aurora B) were fixed and stained with antibodies against XSgo1 (red) and XSgo2 (green). DNA was counterstained with DAPI. Scale bars, 1 μm (insets) and 10 μm. (D) To estimate the efficiency of depletion in the extracts used in (C), 1 μl aliquots were analysed by immunoblotting alongside different amounts of the mock-depleted extract (expressed as percentage of 1 μl). Tubulin was used as a loading control.

XSgo2 does not regulate sister chromatid cohesion. (A) Representative images of replicated mitotic chromosomes assembled in mock-depleted, Δ XSgo1 or Δ XSgo2 extracts that were stained with antibodies against the condensin I subunit XCAP-G (green) and the centromeric histone CENP-A (red). Biotin-dUTP (grayscale images) was added to the extract to monitor DNA replication. Bars, 10 and 1 μm (insets). (B) The distance between sister centromeres was measured for >100 pairs for each condition and shown as the average from three independent experiments. Error bars, s.e.m. (C) Quantitation of the distance between sister chromatids along the length of several chromosomes (n=10 for each condition). Error bars, s.e.m.

Defective spindle assembly in the absence of XSgo2. (A) Immunoblot analysis of extract depletion, as in . (B) Depleted extracts from (A) were cycled into interphase and back into mitosis. Metaphase spindles were assembled for 90 min after CSF addition in the presence of rhodamine-tubulin (red) and DNA was stained with DAPI (blue). The spindle structures found were classified in the categories indicated and quantified for each condition. Data represent mean values (expressed as percentage) obtained from >200 structures in each of three independent experiments. Error bars, s.e.m. (C) Microtubule asters were assembled in mock and Δ XSgo2 mitotic extracts containing rhodamine-labelled tubulin by addition of 10 μM RanQ69L (or buffer as control). Several aliquots of 1 μl were taken, mounted on a slide with fixative solution, and the number of asters in each slide counted and plotted in the bar graph on the right (bars represent mean values, and error bars s.e.m.). Scale bar on images, 10 μm.

XSgo2 is required for MCAK targeting to centromeres and chromosome alignment. (A) Spindles assembled in mock, Δ XSgo1 or Δ XSgo2 cycled extracts containing rhodamine-labelled tubulin were fixed and analysed for immunofluorescence with antibodies against MCAK. DNA was stained with DAPI (blue). (B) Quantification of bipolar spindles with aligned or misaligned chromosomes assembled in the indicated extracts. The data represent the mean from three independent experiments. Error bars, s.e.m. Representative images of each condition are shown (bottom). (C, D) Chromosomes assembled in the indicated mitotic extracts in the presence of nocodazole were immunostained with antibodies against CENP-A and either anti-MCAK (C) or anti-MCAKpS196 (D). DNA was counterstained with DAPI (blue). Quantitation of the fluorescence intensity of anti-MCAK (C) or anti-MCAKpS196 (D) at centromeres and arm regions from at least 10 nuclei per condition in each of three independent experiments is shown on the right. Error bars, s.e.m. Scale bars, 10 μm.

XSgo2 interacts with the CPC and modulates its activity but not its localization. (A) Immunoprecipitation reactions from mitotic extracts using IgG as a control, anti-XSgo1 and two different antibodies against XSgo2, one raised against recombinant protein (XSgo2-rec) and another against a synthetic peptide (XSgo2-tail). (B, C) Mitotic chromosomes were assembled in depleted extracts (as indicated) and immunostained with antibodies against Aurora B and Bub1 (B) or Aurora B-pT248 and the centromeric protein CENP-C (C). DNA was counterstained with DAPI. The fluorescence intensity of Aurora B and Aurora B-pT248 stainings at individual centromeres is plotted. Data come from n>10 nuclei per condition from two experiments.

XSgo2 and Haspin contribute to proper spindle assembly through distinct pathways involving the CPC. (A) Immunoblot analysis of the XSgo2- and Haspin-depleted extracts used for spindle assembly. H3pThr3 is used to measure the efficiency of Haspin depletion whereas a robust non-specific band served as loading control. (B) Spindles assembled in the indicated extracts were classified as in . At least 100 spindle structures were quantified for each condition. A single, representative experiment is shown. (C) Spindle length was measured in at least 25 bipolar spindles assembled in (B). Error bars, s.d.

XSgo1 and XSgo2 interact with different PP2A complexes. (A) Immunoprecipitates obtained from mitotic extracts with control rabbit IgG, anti-XSgo1 and anti-XSgo2 were analysed by immunoblotting. (B) Affinity purification of XSgo2 from mitotic extracts. Input, flow-through (FT) and eluted fractions (E1–E4) were analysed by immunoblotting. In the blot with anti-PP2A-B56ɛ, the arrow indicates the position of the phosphatase subunit and the asterisk a cross-reacting band. Fraction E2 was subjected to mass spectrometry analysis (). (C) In-vitro translated PP2A-B56ɛ tagged with V5 (PP2A-B56ɛ-V5) was added to a mitotic extract and immunoprecipitation reactions were carried out with control IgG, XSgo1 and XSgo2 antibodies. The presence of PP2A-B56ɛ-V5 in the immunoprecipitates was revealed by immunoblot with anti-V5 antibodies (arrow). The asterisk marks the position of IgG heavy chains. Input, 1% of the reaction. (D) Immunoblot analyses of mitotic extracts depleted of PP2A-B56γ, XSgo1 or XSgo2. The asterisk marks a cross-reacting band.

Sgo proteins have non-overlapping functions in chromosome segregation. XSgo1 associates with PP2A-B56γ complex to counteract phosphorylation of cohesin and thereby prevent its dissociation from centromeres until anaphase. XSgo2, in association with PP2A-B56ɛ complex, ensures proper spindle assembly and chromosome congression by promoting proper localization and activity of MCAK. This effect appears to be mediated by the CPC, at least in part. While XSgo1 is required for centromeric accumulation of the CPC, XSgo2 contributes to its activation. In turn, the CPC is required for proper localization of both XSgo1 and XSgo2, and so is Bub1 (not depicted). Green arrows indicate activation; grey arrows indicate regulation of localization. Sgo2 could regulate MCAK not only through the CPC but also in a more direct way (dotted lines).